3.4 Results and discussion
3.4.1.2 Chemosensitivity of the glioblastoma cells against anthracyclines
3.4.1.2.1 Chemosensitivity against doxorubicin
With respect to the investigation of newly developed doxorubicin formulations, the chemosensitivity of human glioblastoma cell lines against non-modified doxorubicin in solution had to be investigated.
Figure 3.3: Effect of dissolved doxorubicin on the proliferation of U-87 MG (a; 136th passage), U-118 MG (b;
455th passage) and U-373 MG (c; 188th passage) cells.
Cells were incubated with: vehicle EtOH 70 % (open stars), 10 nM of vinblastine (open diamonds) and various concentrations of doxorubicin: 3 nM (filled squares), 10 nM (filled circles), 50 nM (filled triangles), 100 nM (filled inverted triangles) or 500 nM (filled diamonds).
Antiproliferative activity of doxorubicin-loaded NPs and doxorubicin derivatives 45
Therefore, the cells were incubated with doxorubicin at increasing concentrations and afterwards processed according to the described procedure (see 3.3.3). Results are shown in Figure 3.3.
As a positive control for assay correctness, 16 wells of the microtiter plate were incubated with 10 nM of vinblastine whereas effects of the solvent were checked by incubation of further 16 wells with the equivalent amount of ethanol 70 %.
Doxorubicin at a concentration of 10 nM caused only a slight cytotoxic effect on all investigated glioblastoma cells. However, incubation of the cells with 100 nM of doxorubicin yielded strong cytotoxic effects on U-87 MG and U-118 MG cells, whereas the same concentration resulted in a cytostatic effect on U-373 MG cells. These data are in good agreement with results of analogous chemosensitivity assays performed by Dr. Peter Altenschöpfer [Altenschöpfer, 1998]. At the highest tested concentration of 500 nM, doxorubicin caused a pronounced cytocidal effect on U-87 MG as well as on U-373 MG cells, whereas a strong cytostatic effect was observed for U-118 MG cells.
3.4.1.2.2 Chemosensitivity against doxorubicin derivatives
2 structural modified doxorubicin derivatives were investigated on their toxicity against U-87 MG, U-118 MG and U-373 MG cells. Basically, these compounds were synthesized with the idea of specifically targeting hormone-dependent breast cancers by addressing the estrogen receptor.
c Figure 3.4: Effect of doxorubicin (filled
symbols) and its derivative Sb-59 (open symbols) on proliferating U-87 MG (a;
136th and 174th passage), U-118 MG (b;
455th and 488th passage) and U-373 MG (c; 188th and 223rd passage) cells
Cells were incubated with the 2 anthracyclines at concentrations of: 10 nM (circles), 100 nM (squares) and 500 nM (triangles).
Antiproliferative activity of doxorubicin-loaded NPs and doxorubicin derivatives 47
Since these compounds show increased lipophilicity compared to doxorubicin, and this might be beneficial to overcome the blood-brain barrier, the derivatives were regarded as promising candidates for preliminary toxicity studies on human glioblastoma cell lines. For this purpose, U-87 MG, U-118 MG and U-373 MG cells were incubated with 3 concentrations of the 2 derivatives, Sb-59 and Sb-63b, respectively using doxorubicin as a reference compound (Figure 3.4 and Figure 3.5).
Figure 3.5: Effect of doxorubicin (filled symbols) and Sb-63b on proliferating U-87 MG (a; 136th and 180th passage), U-118 MG (b; 455th and 495th passage) and U-373 MG (c; 188th and 230th passage) cells.
Cells were incubated with the 2 anthracyclines at concentrations of: 10 nM (circles), 100 nM (squares) and 500 nM (triangles).
At a concentration of 10 nM, Sb-59 had a weak antiproliferative effect on human U-87 MG glioblastoma cells similar to that of 10 nM doxorubicin (Figure 3.4a). However, at concentrations as high as 100 and 500 nM, Sb-59 was less potent than doxorubicin. Whereas 500 nM Sb-59 caused a strong cytostatic effect, the same concentration of doxorubicin was cytocidal.
Similar results were obtained for U-118 MG cells in the presence of doxorubicin and Sb-59 (Figure 3.4b).
Among the 3 human glioblastoma cell types, U-373 MG turned out to be most sensitive against doxorubicin and Sb-59 (Figure 3.4c). Even at the lowest tested concentration of 10 nM, cytotoxic and cytostatic effects were observed. Moreover, incubation of U-373 MG cells with 100 and 500 nM of each compound resulted in distinctive cytostatic and cytocidal impact. Again, as noticed for U-87 MG and U-118 MG cells, doxorubicin was demonstrated to be more potent than its derivative Sb-59.
On U-87 MG cells Sb-63b caused increased antiproliferative activity compared to doxorubicin (Figure 3.5a). At a concentration of 10 nM, doxorubicin was ineffective, whereas Sb-63b mediated pronounced cytostatic effects. Moreover, at higher concentrations (100 and 500 nM), Sb-63b showed increased cytostatic effects compared to doxorubicin, or were even elevated to cytocidal impacts.
Contrarily, doxorubicin was more cytotoxic than Sb-63b against U-118 MG cells (Figure 3.5b), although incubation with 100 and 500 nM of Sb-63b resulted in strong cytostatic and cytocidal effects.
Proliferating U-373 MG cells showed comparable susceptibility against Sb-63b and doxorubicin (Figure 3.5c). Both compounds were cytotoxic at a concentration as low as 10 nM. Whereas at a concentration of 100 nM Sb-63b mediated cytocidal outcomes, doxorubicin caused strong cytostatic effects. In addition, incubation of U-373 MG cells with 500 nM of doxorubicin and Sb-63b, respectively, indicated equi-activity.
Antiproliferative activity of doxorubicin-loaded NPs and doxorubicin derivatives 49
3.4.1.3 Cytotoxicity of different doxorubicin-loaded NP formulations
For investigations on the chemosensitivity of doxorubicin-loaded nanoparticle formulations, the human glioblastoma cells were incubated with the drug-loaded PBCA particles.
Additionally, doxorubicin-loaded PBCA nanoparticles were coated with polysorbate 80 to examine the chemosensitivity of these surface-modified drug-carrier systems.
Figure 3.6 shows the results which were obtained after incubation of U-87 MG, U-118 MG and U-373 MG glioblastoma cells with non-coated doxorubicin-loaded nanoparticles. Similar to investigations performed with doxorubicin in solution, the incubation with 10 nM of doxorubicin-loaded nanoparticles caused only weak cytotoxic effects on both, U-87 MG and U-118 MG cells. Furthermore, the incubation of U-373 MG cells with 10 nM of the 2 different drug formulations (doxorubicin in solution vs. doxorubicin NP, respectively) revealed only a slightly enhanced cytotoxicity of the nanoparticle formulation. The highest tested concentration of doxorubicin-loaded NP (500 nM) resulted in a pronounced cytocidal effect against U-87 MG as well as U-373 MG cells, comparable to outcomes observed for doxorubicin in solution. At the latter concentration (500 nM), the doxorubicin-loaded NP mediated cytocidal effects to U-118 MG cells, whereas doxorubicin in solution caused only cytostatic outcome.
Nevertheless, a drastic increase in toxicity of doxorubicin by loading the drug to PBCA nanoparticles was not observed, whether for U-87 MG and U-118 MG nor for U-373 MG cells.
Overall, the doxorubicin-loaded PBCA nanoparticles proved to be equipotent with the drug in solution. Obviously, the nanoparticle formulation did not enhance the cellular uptake of doxorubicin.